Stroke is the third leading cause of death and the leading cause for long-term disability in the U.S. and other industrialized nations. Each year 750,000 Americans suffer, and more than 150,000 die as a result of new and recurrent strokes. In spite of extensive research efforts, to date only one drug, the thrombolytic agent tPA, has been approved by the FDA for stroke treatment. A number of alternative neuroprotective approaches have been identified in animal ischemia models, but failed in clinical trials. Therefore there is continuous need for development and improvement of novel stroke therapies. In this study we propose to test the novel HYPOTHESIS that oxidative stress potently augments and propagates ischemic brain damage by enhancing glial glutamate release via activation of redox-sensitive glutamate transport pathways. Studies from this and other laboratories has identified three redox-sensitive glutamate release pathways: volume-regulated anion channels (VRAC), the cystine/glutamate antiporter (xCT), and the connexin hemichannels (Cxs). In the proposed work we will use pure and mixed primary cultures of rat astrocytes and microglial cells to find the optimal approaches for reducing pathological glutamate release from glia. This will be followed by testing novel pharmacological treatments in vivo, in a rat model of transient focal ischemia. The following SPECIFIC AIMS are proposed: (AIM 1) to elucidate relative contribution of VRAC, xCT, and Cxs to glutamate release in response to oxidative stress in primary glial cultures, using pharmacological tools and an siRNA approach; (AIM 2) to identify the relationship between oxidative stress and pathological glutamate release in vivo in a rat model of transient focal ischemia, using a microdialysis approach and the intra-dialysate drug delivery; (AIM 3) to test the neuroprotective potential of the pharmacological treatments validated in AIMS 1 and 2, by measuring brain infarction volumes in the control ischemia group and after treatment with neuroprotectants. The major objective of this study is to establish a new fundamental relationship between tissue production of reactive oxygen species and pathological glutamate release from glial cells, and to identify novel pharmacological targets in stroke and other neurological disorders (such as multiple sclerosis, Alzheimer's disease, and traumatic brain injury), which involve oxidative stress.